Mission Bio, Inc. develops instrumentation that will accelerate the investigation of cellular heterogeneity relevant to human health by rapidly and accurately performing single-cell nucleic acid analysis. We previously introduced a new sorting technology, PCR-activated cell sorting (PACS), which is both high in throughput and able to analyze a wide variety of nucleic acid biomarkers with unmatched sensitivity and specificity. Using multiplexed TaqMan PCR assays performed in microfluidic droplets, the system is able to interrogate individual cells for the expression of specific combinations of transcripts, genomic DNA, mutations or non-coding RNAs, targets not amenable to antibody-based detection. While this platform has proven widely useful for identifying and sorting many rare and unique cells, it relies on endpoint PCR reactions and is not able to precisely discriminate between similar cell types possessing only minor differences in nucleic acid copy number. The objective of this SBIR proposal is to develop a platform for performing precise single-cell nucleic acid quantitation on tens of thousands of cells. True quantitative capability with single-cell resolution requires that the TaqMan reaction fluorescence be measured and recorded at the end of each cycle of PCR. To do this with our single-cell droplet reactions, we must build a microfluidic chip, the qCell Array, that can permit thermocycling, fluorescence imaging and spatial tracking of all droplets. The capability provided by such an approach would represent a breakthrough over current methods for quantitative single-cell qPCR that are expensive, labor intensive and/or lack the throughput necessary to analyze large numbers of cells. The intellectual merits of the qCell Array approach stem from the core ability to deliver a rapid and low- cost solution for massively parallel, quantitative single-cell genetic and transcriptional analysis on large heterogeneous populations of cells. The impact of cellular heterogeneity on biological function and disease is crucially important to questions in human immunology, stem cell biology, infectious disease and cancer research. By analyzing individual cells within a mixed population, it is possible to identif rare cell populations or transient cell states critical to human health and development that are otherwise unobservable by ensemble measurements. Commercialization of the qCell Array platform will enable a more detailed and comprehensive analysis of the genes and transcripts in these heterogeneous cell populations. The qCell Array relies on proven molecular biology approaches, advanced microfluidics, and a solid foundation of intellectual property. Phase I funding will result in the development of a needed and technically achievable research and diagnostic tool with high impact potential in the biomedical marketplace.